Lipid rafts play an important role in signal integration and cellular activation of a number of cytokine and growth factor receptors. Flotillin proteins have recently been shown to be recruited to lipid raft microdomains upon cellular activation and have been implicated in neural cell regeneration, receptor signaling and lymphocyte activation. However, little is known about the relevance of the flotillin proteins in T cell responses to chemoattractant stimulation. To this end, cytoplasmic and lipid raft fractions from human T cells were analyzed for flotillin protein redistribution prior to and after CXCL12 stimulation. Flotillin-1 but not flotillin-2 redistributes to lipid rafts upon CXCR4 ligation. Moreover, in CXCL12-treated T cells, flotillin-1 also associates with several raft proteins including LAT, Lck, CD48 and CD11a. In addition, an increase in CXCR4 association with flotillin-1 in lipid rafts was observed after chemokine treatment. RNAi technology was also utilized to inhibit the expression of flotillin-1 resulting in an inhibition of CXCL12-mediated signaling, function and CXCR4 recruitment into lipid rafts. Together, these data suggest that the association of flotillin-1 with lipid raft during chemokine exposure may play an important role in chemokine receptor recruitment to and signaling in lipid rafts and possibly in leading edge formation. Overall, we believe that a greater understanding of the various signaling and cell surface proteins associated with lipid rafts may provide insight into age-related alterations in cell signaling and trafficking.